DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs

Abstract

RNA silencing is a conserved eukaryotic gene expression regulatory mechanism mediated by small RNAs. In Caenorhabditis elegans, the accumulation of a distinct class of siRNAs synthesized by an RNA-dependent RNA polymerase (RdRP) requires the PIR-1 phosphatase. However, the function of PIR-1 in RNAi has remained unclear. Since mammals lack an analogous siRNA biogenesis pathway, an RNA silencing role for the mammalian PIR-1 homolog (dual specificity phosphatase 11 [DUSP11]) was unexpected. Here, we show that the RNA triphosphatase activity of DUSP11 promotes the RNA silencing activity of viral microRNAs (miRNAs) derived from RNA polymerase III (RNAP III) transcribed precursors. Our results demonstrate that DUSP11 converts the 5 ′ triphosphate of miRNA precursors to a 5 ′ monophosphate, promoting loading of derivative 5p miRNAs into Argonaute proteins via a Dicer-coupled 5 ′ monophosphate-dependent strand selection mechanism. This mechanistic insight supports a likely shared function for PIR-1 in C. elegans. Furthermore, we show that DUSP11 modulates the 5 ′ end phosphate group and/or steady-state level of several host RNAP III transcripts, including vault RNAs and Alu transcripts. This study shows that steady-state levels of select noncoding RNAs are regulated by DUSP11 and defines a previously unknown portal for small RNA-mediated silencing in mammals, revealing that DUSP11-dependent RNA silencing activities are shared among diverse metazoans.

Y-RNAs Lead an Endogenous Program of RIG-I Agonism Mobilized upon RNA Virus Infection and Targeted by HIV

“…We generated DUSP11 knock-out Jurkat cells to analyze its activity on Y-RNAs. Deletion of DUSP11 led to a notable increase in 5’-PPP levels at RNY4 5’-end compared to WT cells, as previous results suggested ( 20 ) (Fig. S3B, S3C).…”

“…S4). While we cannot exclude that other functions associated with DUSP11, such as the maturation of miRNA ( 20, 21 ), might explain the selection forces leading to its targeting by HIV-1, we can also speculate that RLR activation by Y-RNAs may act as a rapid response mechanism for hosts to detect viruses that degrade DUSP11. Importantly, during the chronic phase of HIV-1 infection, higher levels of IFN-I signaling correlate with sustained levels of inflammation, immune exhaustion, CD4 T cell depletion and disease progression ( 28 ).…”

“…Every Pol3-transcribed RNA initially contains a 5’-PPP upon transcription that can be further edited by different cellular enzymes. Among these, DUSP11 is a protein from the dual-specificity phosphatase family that displays 5’-triphophatase activity on several miRNA and other cellular noncoding RNAs ( 19, 20 ). We generated DUSP11 knock-out Jurkat cells to analyze its activity on Y-RNAs.…”

Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction

“…An attractive feature of the TGIRT Total RNA-seq method is that it enables the comprehensive analysis of different RNA size classes in a single RNA-seq experiment, enabling applications such as comparison of mRNA codon usage with cellular tRNA levels (Bazzini et al 2016;Smith et al 2018), co-expression of small ncRNAs and mRNAs encoding components of RNP complexes (Boiven et al 2018), and analysis of tRNAs and tRNA fragments or mature, pre-, and pri-miRNA in the same RNA-seq Qin et al 2016;Burke et al 2016;Wang et al 2018). Previous work showed that the total RNA-seq protocol with TGIRT-III works well for quantitation of small RNAs down to ~60 nt (Boivin et al 2018), and the introduction of the NTT adapter in the present work substantially improves the representation of miRNAs in the datasets.…”

Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes

“…Bovine leukemia virus (BLV) expresses five pre-miRNAs from ~550 bps of genomic space (12). Each pre-miRNA is directly transcribed by RNAP III from individual compact RNAP III type II genes (13)(14)(15). This is analogous to typical shRNA generation, but unlike the U6/H1 promoters, the two cis promoter elements (the A and B boxes) that promote RNAP III transcription initiation are located within or directly downstream of the pre-miRNA hairpin (13,16,17).…”